title: A new human primary epithelial cell culture model to study conjunctival inflammation.
abstract: OBJECTIVE
  To develop a complete and optimized method to expand in culture human conjunctival epithelial cells from cadaveric donor samples.
  
  
  METHODS
  Epithelial cells were obtained from cadaveric conjunctival tissue (n = 47). Preplating and differential trypsinization were optimized to eliminate stromal contamination. Epithelial cells were grown with five different media: control, epithelial growth factor (EGF)-enriched, H2O2-supplemented, fibroblast-conditioned, and human serum media. Adhesion, proliferation, colony forming efficiency (CFE), and percentage of CK19(+) and Ki67(+) cells were determined with the five different media. Cells were characterized by immunofluorescence and/or Western blotting techniques for the expression of CK4, CK7, CK19, MUC5AC, vimentin, FSP-1, Ki67, E-cadherin, and zonula occludens (ZO)-1 markers. In addition, cells were treated with TNF-α and levels of secreted IL-6 were measured by enzyme-linked immunosorbent assay.
  
  
  RESULTS
  Pure epithelial cell cultures were obtained. Human serum medium showed the best properties in proliferation and CFE, while maintaining epithelial phenotype. Cells with this medium were passaged up to five times, although they maintained all epithelial characteristics only through passage 3. Cultured cells expressed epithelial markers, but not stromal ones. The number of MUC5AC(+) cells increased throughout the passages, whereas Ki67(+) cell numbers decreased. Cells in culture maintained adherens and tight junctions, and responded to TNF-α treatment by releasing more IL-6, showing that they can be used for inflammation assays.
  
  
  CONCLUSIONS
  We have developed a complete protocol to expand conjunctival epithelial cells from cadaveric tissue. This culture system responded to an inflammatory stimulus, so it could be used to develop a more complex in vitro model of inflammation.
authors.affiliation: Ocular Surface Group, Institute for Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: García-Posadas
authors.forename: Isabel
authors.initials: I
authors.lastname: Arranz-Valsero
authors.forename: Antonio
authors.initials: A
authors.lastname: López-García
authors.forename: Laura
authors.initials: L
authors.lastname: Soriano-Romaní
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Diebold
mesh_terms: D000704:Analysis of Variance; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western; D002102:Cadaver; D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D003228:Conjunctiva / Q000166:cytology*; D003231:Conjunctivitis / Q000473:pathology*; D003470:Culture Media / Q000737:chemistry; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D007150:Immunohistochemistry; D015850:Interleukin-6 / Q000378:metabolism; D008954:Models, Biological; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D015415:Biomarkers; D003470:Culture Media; D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha
keywords: cadaveric donor; cell culture; conjunctival epithelial cells